Saccharopolyspora culture

ABSTRACT

A process for preparing the antibiotic U-59,760. The process is a microbiological process using a biologically pure culture of the novel microbe Saccharopolyspora hirsuta strain 367, NRRL 12045. The antibiotic produced by the process is active against various microorganisms, for example, Staphylococcus aureus, Streptococcus pyogenes, and Klebsiella pneumoniae. Thus, antibiotic U-59,760 can be used in various environments to eradicate or control such bacteria.

This is a division of application Ser. No. 081,211, filed Oct. 2, 1979, now U.S. Pat. No. 4,293,651.

BRIEF SUMMARY OF THE INVENTION

The process of the invention utilizes the biologically pure culture of the novel microbe Saccharopolyspora hirsuta strain 367, NRRL 12045, to produce the useful antibiotic U-59,760. The physical and chemical characteristics of this antibiotic cannot be distinguished from the antibiotic known as compound 47444 which is disclosed in U.S. Pat. No. 4,148,883. The production of the antibiotic in U.S. Pat. No. 4,148,883 is accomplished by an entirely different microbe which is classified as a Nocardia.

DETAILED DESCRIPTION OF THE INVENTION

The microorganism used for the production of antibiotic U-59,760, as described herein, is a biologically pure culture of Saccharopolyspora hirsuta strain 367, NRRL 12045.

A subculture of this microorganism can be obtained from the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., U.S.A. Its accession number in this depository is NRRL 12045. It should be understood that the availability of the culture does not constitute a license to practice the subject invention in derogation of patent rights granted with the subject instrument by governmental action.

The microorganism of this invention was studied and characterized by Alma Dietz and Grace P. Li of The Upjohn Company Research Laboratories.

An actinomycete isolated in the Upjohn soils screening laboratory has been characterized and found to have the macroscopic, microscopic and whole cell hydrolysate properties of the genus Saccharopolyspora [Iwasaki, A., N. Itoh, and T. Mori, 1979. A new broad-spectrum aminoglycoside antibiotic complex, sporaricin. II. Taxonomic studies on the sporaricin producing strain Saccharopolyspora hirsuta subsp. kobensis nov. subsp. J. Antibiotics. 32: 180-186.] [Lacey, J. and M. Goodfellow. 1975. A novel actinomycete from sugar-cane bagasse: Saccharopolyspora hirsuta gen. et sp. novl. J. Gen. Microbiol. 88: 75-87]. A comparison with the type culture Saccharopolyspora hirsuta ATCC 27875 is given in Tables 1 and 2. A comparison with the type culture and with the subspecies, Saccharopolyspora hirsuta ss. kobensis ATCC 20501 [Iwasaki, A. supra] based on data published for these strains is given in Table 3.

Unique properties of the genus Saccharopolyspora are its butyrous or gelatinous-type vegetative growth, sparse aerial growth which is best studied after 21 days incubation, yellow to orange to orange-tan vegetative growth and pigment production, spore chains developing in a sheath with unique hair tufts with truncated bases and smooth areas between the tufts, the presence of meso-diaminopimelic acid, arabinose and galactose in whole cell hydrolysates and in cell wall preparations. L-diaminopimelic acid was detected in addition to meso-DAP in our cell wall preparations.

No antibiotic production has been reported for the type culture; the subspecies kobensis produces the antibiotic complex sporaricin (KA-6606) [Iwasaki, A. supra]. The new isolate is distinguished by the production of antibiotic U-59,760 and U-59,761. The only significant difference among the strains is in their antibiotic production capability. Therefore, the new strain is designated Saccharopolyspora hirsuta strain 367.

The taxonomic methods used herein were those cited in Dietz [Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete classification. Ann N.Y. Acad. Sci. 60: 152-154.] [Dietz, A. 1967. Streptomyces steffisburgensis sp. n. J. Bacteriol. 94: 2022-2026.], Dietz and Mathews [Dietz, A., and J. Mathews. 1971. Classification of Streptomyces spore surfaces into five groups. Appl. Microbiol. 21: 527-533.], Becker et al. [Becker, B., M. P. Lechevalier, and H. A. Lechevalier. 1966. Chemical composition of cell wall preparations from strains of various form-genera of aerobic actinomycetes. Appl. Microbiol. 13 236-243.], Lechevalier and Lechevalier [Lechevalier, H. A., and M. P. Lechevalier. 1970. A critical evaluation of the genera of aerobic actinomycetes, p. 393-405. In H. Prauser (ed.), The Actinomycetales. Gustav Fisher, Jena.] [Lechevalier, M. P., and H. A. Lechevalier. 1970. Chemical composition as a criterion in the classification of aerobic actinomycetes. Int. J. Syst. Bacteriol. 20: 435-443.], and Shirling and Gottlieb [Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16: 313-340.].

Color characteristics: Aerial mycelium gray to gray-pink (very sparse at 14 days). Melanin negative. Appearance on Ektachrome is given in Table 1. Surface colors of the vegetative growth were determined to be red (R) or yellow (Y) by matching the color with the chips in the Tresner and Backus [Tresner, H. D., and E. J. Backus. 1963. System of color wheels for streptomycete taxonomy. Appl. Microbiol. 11: 335-338.] color wheels.

Microscopic characteristics: Spore masses and spore chains, intact and fragmented, and flexuous or loosely spiraled chains, as observed by Scanning Electron Microscopy, appear to develop in a sheath which is at first almost smooth; then unique tufts of hairs (triangular at the base) are formed. The surface between the tufts is smooth. Finally, the sheath becomes covered with fine long hairs. The long sheaths are tangled and appear to arise from, or fuse to form pseudosporangia. As the chains come in contact with the substrate the spores become smooth in outline with a depressed or ridged appearance. "Empty" hyphae are noted in the developed chains. Microscopic characteristics are best noted after 21 days incubation at 28°-37° C. Nocardioform fragmentation may be seen in 21 day cultures.

Growth on carbon compounds: Under the test conditions of Shirling and Gottlieb [supra] growth was good on the postive control (basal medium plus D-glucose), sucrose, D-mannitol, D-fructose and raffinose. Growth was doubtful on L-arabinose and D-xylose. There was no growth on the negative control (basal medium only), inositol, rhamnose, and cellulose.

Whole cell analysis: meso-Diaminopimelic acid, arabinose and galactose were detected.

Cell wall analysis: meso-Diaminopimelic acid, L-diaminopimelic acid, arabinose and galactose were found.

Cultural and biochemical characteristics: See Table 2.

Temperature: There was no to very poor growth at 18° C. on Bennett's and maltose-tryptone agars and poor growth on Czapek's sucrose agar. Growth was good at 24°-45° C. There was no growth at 55° C.

                                      TABLE 1                                      __________________________________________________________________________     Color characteristics* of Saccharopolyspora hirsuta ATCC 27875 and             S. hirsuta strain 367 on Ektachrome                                                             S. hirsuta ATCC 27875                                                                         S. hirsuta strain 367                          Agar Medium                                                                             Determination                                                                          Chip                                                                              Color       Chip                                                                              Color                                       __________________________________________________________________________     Bennett's                                                                               S       71 moderate orange yellow                                                                     71 moderate orange yellow                               R       71 moderate orange yellow                                                                     71 moderate orange yellow                      Czapek's sucrose                                                                        S       74 strong yellowish brown                                                                     67 brilliant orange yellow                              R       72 dark orange yellow                                                                         67 brilliant orange yellow                     Maltose-tryptone                                                                        S       71 moderate orange yellow                                                                     71 moderate orange yellow                               R       71 moderate orange yellow                                                                     71 moderate orange yellow                      Peptone-iron                                                                            S       68 strong orange yellow                                                                       68 strong orange yellow                                 R       68 strong orange yellow                                                                       68 strong orange yellow                        0.1% Tyrosine                                                                           S       73 pale orange yellow                                                                         71 moderate orange yellow                               R       73 pale orange yellow                                                                         71 moderate orange yellow                      Casein   S       73 pale orange yellow                                                                         70 light orange yellow                                  R       73 pale orange yellow                                                                         73 pale orange yellow                          __________________________________________________________________________      *Color was determined by comparison with NBS color chips (SP 440. Color:       Universal Language and Dictionary of Names. U.S. Government Printing           Office, Washington, DC 20402. SRM 2106. ISCCNBS Centroid Color Charts.         Office of Standard Reference Material, Room B311, Chem. Building, Nationa      Bureau of Standards, Washington, DC 20234. S = Surface R = Reverse       

                                      TABLE 2                                      __________________________________________________________________________     Cultural and biochemical characteristics of Saccharopolyspora hirsuta          strains.                                                                       Agar medium                                                                              Determination                                                                          S. hirsuta ATCC 27875                                                                       S. hirsuta strain 367                           __________________________________________________________________________     Peptone-iron                                                                             S       Colorless (V)                                                                               Colorless or light tan                                                         wrinkled (V)                                              R       Pale yellow  Orange-tan                                                P       --           Pale tan to orange-tan                                    O       Melanin negative                                                                            Melanin negative                                Calcium malate                                                                           S       Trace white (A)                                                                             Trace gray (A)                                            R       Cream        Pale gray cream                                           P       --           --                                                        O       Malate solubilized                                                                          Malate slightly solubilized                     Glucose asparagine                                                                       S       Colorless to light tan (V)                                                                  Light tan (V)                                             R       Orange       Orange-tan                                                P       Orange       Orange-tan                                      Skim milk S       Tan (V)      Orange-brown (V)                                          R       Orange-tan   Dark orange-tan                                           P       Orange-tan   Deep orange-tan                                           O       Casein solubilized                                                                          Casein solubilized                              Tyrosine  S       Tan (V)      Orange-tan (V)                                            R       Orange       Orange                                                    P       Orange       Orange                                                    O       Tyrosine solubilized                                                                        Tyrosine solubilized                            Xanthine  S       Colorless (V)                                                                               Pale tan (V)                                              R       Pale yellow  Pale tan                                                  P       Pale yellow  Pale tan-peach                                            O       Xanthine solubilized                                                                        Xanthine solubilized                                              around growth                                                Nutrient starch                                                                          S       Colorless (V)                                                                               Pale tan (V)                                              R       Pale yellow  Pale tan                                                  P       Very pale yellow                                                                            Very pale tan                                             O       Starch hydrolyzed                                                                           Starch hydrolyzed                               Yeast extract-malt                                                                       S       Colorless (V)                                                                               Pale tan (V)                                    extract   R       Orange       Pale-orange-tan                                           P       Orange       Orange                                          Peptone-yeast                                                                            S       Colorless (V) with very                                                                     Colorless (V)                                   extract-iron      slight trace white (A)                                       (ISP-6)   R       Yellow-tan   Light orange-tan to tan                                   O       Melanin negative                                                                            Melanin negative                                Tyrosine (ISP-7)                                                                         S       Trace white (A)                                                                             Very pale orange (V)                                      R       Yellow tan   Very pale orange                                          O       Melanin negative                                                                            Melanin negative                                Gelatin                                                                        Plain     S       Trace white (A) on                                                                          Colorless to pale yellow (V)                                      colorless surface                                                              pellicle                                                               P       None         None to trace pale yellow                                 O       Gelatin liquefied                                                                           Gelatin liquefied                               Nutrient  S       Trace white (A) on                                                                          Trace colorless (V) to trace                                      yellow (V) pellicle                                                                         white (A) on (V)                                          P       None         None                                                      O       Gelatin liquefied                                                                           Gelatin liquefied                               Broth                                                                          Synthetic nitrate                                                                        S       No surface growth                                                                           Trace white (A) on colorless (V)                          P       None         None                                                      O       Compact bottom growth                                                                       Compact to flocculent bottom                                      No reduction growth                                                                         No reduction                                    Nutrient nitrate                                                                         S       Trace white (A) on color-                                                                   Trace white (A) on colorless                                      less surface pellicle                                                                       surface ring                                              P       None         None                                                      O       Flocculent bottom growth;                                                                   Poor colorless compact bottom                                     No reduction growth; No reduction                            Litmus milk                                                                              S       Pale gray (A) on yellow-                                                                    Blue-gray surface ring                                            tan surface ring                                                       P       None         Deep red-tan                                              O       Litmus not reduced                                                                          Peptonization                                                     pH 6.7-7.3   pH 7.6-7.8                                      __________________________________________________________________________      S = Surface                                                                    O = Other characteristics                                                      R = Reverse                                                                    (V) = Vegetative Growth                                                        P = Pigment                                                                    (A) = Aerial growth                                                      

                                      TABLE 3                                      __________________________________________________________________________     Comparision of Saccharopolyspora hirsuta strains                               Tests        S. hirsuta strain 367                                                                     S. hirsuta ATCC 27875                                                                      S. hirsuta kobense ATCC                    __________________________________________________________________________                                         20501                                      Physiological Properties                                                       Temperature  Growth from 18-45C                                                                        Growth from 25-45C                                                                         Growth from 18-45C                         Optimum Temperature                                                                         28-45C     37-40C      37-42C                                     Gelatin liquefaction                                                                        Complete   Positive at 27C                                                                            Positive at 27C                            Starch hydrolysis                                                                           Positive   Positive    Positive                                   Action on milk                                                                              No coagulation                                                                            No coagulation                                                                             No coagulation                                          Peptonization                                                                             Peptonization                                                                              Peptonization                              Melanoid pigment                                                                            Negative   Negative    Negative                                   production                                                                     Nitrate reduction                                                                           Negative   Negative    Positive                                   Carbon Source Utilization                                                      L-Arabinose  ±       -           -                                          D-Xylose     ±       +           -                                          L-Rhamnose   -          +           -                                          D-Glucose    +          +           +                                          D-Fructose   ++         +           +                                          Sucrose      +          +           +                                          Raffinose    ++         +           +                                          Inositol     -          +           -                                          D-Mannitol   ++         +           +                                          __________________________________________________________________________

The compound of the invention process is produced when the elaborating organism is grown in an aqueous nutrient medium under submerged aerobic conditions. It is to be understood, also, that for the preparation of limited amounts surface cultures and bottles can be employed. The organism is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewer's yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers' solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added to the fermentation media since tap water and unpurified ingredients are used as components of the medium prior to sterilization of the medium.

Production of the compound by the invention process can be effected at any temperature conducive to satisfactory growth of the microorganism, for example, between about 18° and 40° C., and preferably between about 20° and 28° C. Ordinarily, optimum production of the compound is obtained in about 3 to 15 days. The final pH of the fermentation is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium.

When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the production of the compound and the attendant inefficient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil, liquid N₂ agar plug, or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of the compound, so long as a good growth of the microorganism is obtained.

A variety of procedures can be employed in the isolation and purification of the compound produced by the subject invention from fermentation beers. Isolation can be accomplished by extraction with solvents such as methylene chloride, acetone, butanol, ethyl acetate and the like; and silica gel chromatography can be used to purify crude preparations of the antibiotic.

In a preferred recovery process, the compound produced by the subject process is recovered from the culture medium by separation of mycelia and undissolved solids by conventional means, such as by filtration or centrifugation, and solvent extraction of both mycelial cake or clarified broth. The clarified broth can be extracted with a suitable solvent, for example, methylene chloride (preferred), ethylacetate, butanol, and MIBK. The extract can be evaporated under reduced pressure to a concentrate and the antibiotic recovered from this concentrate by subjecting the concentrate to chromatography over silica gel and eluting with methanol and methylene chloride.

The following examples are illustrative of the process of the invention, but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1

Part A. Fermentation

A biologically pure culture Saccharopolyspora hirsuta strain 367, NRRL 12045, is used to inoculate 500-ml Erlenmeyer seed flasks containing 100 ml of sterile medium consisting of the following ingredients:

    ______________________________________                                         Glucose monohydrate  25 g/l                                                    Pharmamedia*         25 g/l                                                    Tap water q.s.        1 liter                                                  ______________________________________                                          *Pharmamedia is an industrial grade of cottonseed flour produced by            Traders Oil Mill Company, Fort Worth, Texas.                             

The seed medium presterilization pH is 7.2. The seed inoculum is grown for three days at 28° C. on a Gump rotary shaker operating at 250 rpm and having a 21/2 inch stroke.

After three days incubation, the seed medium is used to inoculate (the inoculation rate is 5 ml of seed inoculum per 100 ml of fermentation medium) a series of 500-ml Erlenmeyer flasks containing sterile fermentation medium consisting of the following ingredients:

    ______________________________________                                         Glucose monohydrate 10       g/l                                               Dextrin             20       g/1                                               Corn steep liquor   2.5      g/l                                               NH.sub.4 NO.sub.3   3.0      g/l                                               NaCl                2.0      g/l                                               CaCO.sub.3          5.0      g/l                                               ph-7.2 (presterilization)                                                      ______________________________________                                    

The fermentation flasks are incubated at a temperature of 28° C. on a Gump torary shaker operating at 250 rpm nd having a 21/2 inch stroke. Harvest is usually after about 5 days of fermentation. A typical 5 day fermentation has the following titers of antibiotic in the fermentation broth:

    ______________________________________                                         Day         S. lutea Assay, Bu/ml                                              ______________________________________                                         2            8.0                                                               3           10.4                                                               4           10.4                                                               5           11.2                                                               ______________________________________                                    

In the assay results, a biounit (BU) is defined as the concentration of the antibiotic which gives a 20 mm zone of inhibition under the standard assay condition. Thus, if, for example, a fermentation beer has to be diluted 1/100 to give a 20 mm zone of inhibition, the potency of such beer is 100 Bu/ml.

B. Recovery and Purification

The whole beer (ca. 5,000 l) from a fermentation, as described above, is adjusted to pH 7.3 with NaOH and filtered on a 30 inch filter press using diatomaceous earth as a filter aid. During the filtration operation wash water is applied to the filter cake. From the filtration operation is recovered 5,500 l of clear fermentation broth which is then extracted twice with methylene chloride (1,400 l each time) to give a total of 2.800 l of solvent extract. This solvent extract is concentrated in vacuo to 10 l. Assay on a standard S. lutea disc plate assay gives a value of 2424 Bu/ml.

The extract concentrate described above (9 l), is chromatographed over a column containing 9 kg of silica gel (E. Merck-silica gel 7734). The column is eluted as follows:

20 liters methylene chloride; then 40 liters 2% methanol in methylene chloride; then 150 liters 5% methanol in methylene chloride; then 100 liters 10% methanol in methylene chloride. Four liter fractions are collected after an 80 liter forerun. Fractions 5-8 contain antibiotic U-59,760 having the following characteristics:

Elemental Analysis: C, 64.95, H, 7.42, N, 2.49

Molecular Weight: 515.2482 (Mass spectroscopy) (C₂₈ H₃₇ NO₈).

Optical Rotation: [a]_(D) ²⁵ +53° (C, 0.0848 MeOH).

Solubilities: Soluble in methanol, ethanol and methylene chloride, and relatively insoluble in petroleum ether and cyclohexane.

Infrared Absorption Spectrum: Antibiotic U-59,670 has a characteristic infrared absorption spectrum in a mineral oil mull. Peaks are observed at the following wave lengths expressed in reciprocal centimeters.

    ______________________________________                                         Band Frequency                                                                 (Wave Numbers)     Intensity                                                   ______________________________________                                         3459               S                                                           3330               S, sh.                                                      3139               M, sh.                                                      3025               M                                                           2957               S                                                           2922               S                                                           2855               S                                                           2725               W                                                           1725               S, sh.                                                      1704               S                                                           1688               S                                                           1554               M                                                           1455               S                                                           1411               S                                                           1377               S                                                           1354               S                                                           1339               S                                                           1315               S, sh.                                                      1307               S                                                           1276               S                                                           1265               S, sh.                                                      1240               S                                                           1205               M                                                           1169               S                                                           1109               S                                                           1082               S                                                           1060               S                                                           1045               S                                                           1031               S                                                           1010               S                                                           968                S                                                           956                S                                                           949                S                                                           937                M                                                           909                M                                                           882                M                                                           870                M, sh.                                                      857                M                                                           835                M                                                           794                M                                                           764                S                                                           747                S                                                           719                M                                                           699                M                                                           677                M                                                           638                M                                                           ______________________________________                                          Key: S = Strong, M = Medium, W =0 Weak, sh. = shoulder                   

    ______________________________________                                         Antimicrobial spectrum:                                                                            Inhibition                                                 Organism            Zone Size (mm).sup.1                                       ______________________________________                                         B. subtilis UC 564  0                                                          B. subtilis UC 6033 16                                                         S. aureus UC 70     36                                                         S aureus UC 3665    42                                                         S. aureus UC 6029   38                                                         S. lutea UC 130     42                                                         S. lutea UC 3383    41                                                         S. lutea sens       45                                                         K. pneumoniae UC 58 32                                                         E. coli UC 51       0                                                          S. schottmuelleri UC 126                                                                           0                                                          P. vulgaris UC 93   0                                                          P. aeruginosa UC 95 0                                                          M. avium UC 159     30                                                         P. oxalicum UC 1268 0                                                          S. pastorianus UC 1342                                                                             0                                                          R. sphaeroides UC 3238                                                                             21                                                         S. pyogenes UC 6055 16                                                         B. fragilis UC 6513 37                                                         C. perfringens UC 6509                                                                             48                                                         ______________________________________                                    

"UC" is a registered trademark of The Upjohn Company culture collection. These cultures can be obtained from The Upjohn Company in Kalamazoo, Mich., upon request.

Since antibiotic U-59,760 is active against S. aureus, it can be used to disinfect washed and stacked food utensils contaminated with this bacteria. Also, it can be used as a disinfectant on various dental and medical equipment contaminated with S. aureus. Further, antibiotic U-59,760 can be used as a bacteriostatic rinse for laundered clothes, and for impregnating papers and fabrics; and, it is also useful for suppressing the growth of sensitive organisms in plate assays, and other microbiological media. 

We claim:
 1. A biologically pure culture of the microorganism Saccharopolyspora hirsuta strain 367, having the identifying characteristics of NRRL 12045, said culture being capable of producing the antibiotic U-59,760 in a recoverable quantity upon fermentation in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances. 